Dissolved fraction characterization

Although the investigations of both Raunkjaer et al. (1995) and Almeida (1999) showed that removal of COD — measured as a dissolved fraction — took place in aerobic sewers, a total COD removal was more difficult to identify. From a process point of view, it is clear that total COD is a parameter with fundamental limitations, because it does not reflect the transformation of dissolved organic fractions of substrates into particulate biomass. The dissolved organic fractions (i.e., VFAs and part of the carbohydrates and proteins) are, from an analytical point of view and under aerobic conditions, considered to be useful indicators of microbial activity and substrate removal in a sewer. The kinetics of the removal or transformations of these components can, however, not clearly be expressed. Removal of dissolved carbohydrates can be empirically described in terms of 1 -order kinetics, but a conceptual formulation of a theory of the microbial activity in a sewer in this way is not possible. The conclusion is that theoretical limitations and methodological problems are major obstacles for characterization of microbial processes in sewers based on bulk parameters like COD, even when these parameters are determined as specific chemical or physical fractions. 

For these reasons, numerous attempts have been made to identify and characterize DOP, but with little success because it is usually present in very low concentrations. Typical values in lake waters range from 5 to 100 xg of P/L in oligotrophic to eutrophic systems. Colorimetric methods have been used extensively to detect and differentiate between soluble reactive phosphorus (SRP) and soluble unreactive phosphorus (SUP) at concentrations as low as 10 xg of P/L (I). SRP is generally considered to consist of only orthophosphate compounds, whereas SUP is composed of all other phosphorus species, primarily organic phosphorus compounds. The sum of SRP and SUP is equal to the total soluble phosphorus (TSP). These methods were used to study the dynamics of bulk phosphorus fractionation between the sediments, suspended particulate matter, the biota, and the dissolved fraction (2). Despite these studies, very little is known regarding the identity and characteristics of the DOP in the hydrosphere. 

FIGURE 12.15 Time series of C/N ratios in the dissolved fraction and in the particulate fraction in the near-bottom layer of station 271. Oxygen concentrations characterizing the redox state are added. IlnS was converted into negative oxygen equivalents. (Updated from Nausch et al., 2005.)... 

Characterization of Dissolved Fraction. The dissolved organic fraction is separated from the suspended organic matter by Millipore filtration using a 0.45-/ m filter, Type HA. The TOC of the filtrate is determined first. Thereafter, the filtrate that contained the dissolved organic matter is separated from the water by chloroform extraction or by means of adsorption on Amberlite XAD-type resin. 

Traditionally, phosphorus research in the water column has been limited to measuring concentrations of operationally defined, but not chemically well-characterized, phosphorus fractions. Aquatic phosphorus is traditionally divided into particulate and dissolved fractions by filtration, with further division into phosphate, inorganic condensed, organic condensed, organic, mineral, and mixed-phase phosphorus species (Holtan et al., 1988 Maher and Woo, 1998, and references therein see also McKelvie, Chapter 1 Cade-Menun, Chapter 2 Cooper et al.. Chapter 3, this volume). 

Preparative fractionation of lOOmg sample of HPD and the product of the DP-HP synthesis were carried out on a 5x30cm Sephadex LH-20 column (Pharmacia, Piscataway, NJ.). The porphyrin components were first dissolved in 40ml of tetrahydrofuran-methanol-5mM aqueous phosphate buffer, pH 7, (2 1 1), and subsequently eluted with the same solvent. The use of this methodology for the isolation of the tumor-localizing fraction of HPD from other porphyrin components present in the drug mixture has been discussed in detail in the literature.(11) The component of HPD which elutes very close to the void volume of the LH-20 column has been characterized as the tumor-localizing fraction of HPD. 

Compound 8 (40 mg) was dissolved in DMF (300 pL), FITC (40 mg) was added, and the mixture adjusted to pH 8.0 with NMM. After incubation for 15 h in the dark, the product was purified by chromatography (silica) on a Kieselgel 60 1.5 x 20-cm column (Fluka Chemie, Buchs, Switzerland) equilibrated in MeOH/CHCl3 (1 1). The excess FITC eluted in the flow-through fraction, and a second yellow fraction containing the expected product was eluted with MeOH/CHCl, (4 1). The solvent was removed by rotary evaporation, and the product was characterized by ESI-MS mlz. [M+H]+ calcd, 709.6 found, 709.4. Further purification was by semipreparative RP-HPLC on a Nucleosil-C8 column (10 x 250 mm) at a flow rate of 4mL min 1 using a 30-60% gradient (same components as above) over 30 min. 

An equimolar mixture of Z(OMe)-Cys(Mob)-Gly-OBzl (0.1 mmol) and Z(OMe)-Cys(Mob)(0)-Ala-NH2 (0.1 mmol) was treated with 1M TfOH/TFA (3 mL) containing Me2S (0.5 M) in an ice-bath for 1 h, then hexane was added. The resulting precipitate was dissolved in H20 and the aqueous phase was washed with Et20. The soln was treated with Amberlite CG-4B (acetate form) for 30 min then the resin was removed by filtration. The filtrate was applied to a column of Sephadex G15 which was eluted with 1M AcOH, the fractions corresponding to the front main peak were combined and lyophilized. The residue was purified by HPLC to give the title product yield 87% [a]D22 —60.9 (c 0.2, H20). The product was further characterized by FAB-MS and amino acid analysis. 

HPLC. The crude product was dissolved in 0.1% aq TFA (10 mL), filtered, and purified by preparative HPLC [Preppak, Cartridge YMC ODS column (4.8 x 30 cm), gradient elution MeCN/0.1% TFA/H20 from 20 to 50% MeCN in 90 min (flow rate of 50mL-min-1)]. The fractions containing pure product were combined, concentrated, and lyophilized yield 22.4mg (44%) the peptide was characterized by HPLC (purity 98%), amino acid analysis, and FAB-MS. 

The discovery happened by accident. Lewis and Anders were frustrated by their failure to find the carrier of anomalous xenon in carbonaceous chondrites. They decided to try an extreme treatment to see if they could dissolve the carrier. They treated a sample of the colloidal fraction of an Allende residue with the harshest chemical oxidant known, hot perchloric acid. The black residue turned white, and to their surprise, when they measured it, the anomalous xenon was still there The residue consisted entirely of carbon, and when they performed electron diffraction measurements on it, they found that it consisted of tiny (nanometer sized) diamonds. After a detailed characterization that included chemical, structural, and isotopic studies, they reported the discovery of presolar diamond in early 1987 (Lewis et al., 1987). The 23-year search for the carrier of CCFXe (Xe-HL) was over, and the study of presolar grains had begun. 

Incubation of lake water with 32P or 33P as tracers and subsequent gel chromatography reveals that a major pathway exists between dissolved orthophosphate and the particulate phase (3, 5-7). Low-molecular-weight phosphorus forms in the presence of bacteria and algae. SUP is present in the low-molecular-weight fraction and is classified as individual DOP compounds unassociated with particulate or colloidal material. The HMW fraction found in gel chromatography studies is characterized as a colloid that contains phosphorus compounds or incorporates orthophosphate. The colloidal material then releases orthophosphate, replenishing the dissolved phosphorus cycle. In some eutrophic lakes the HMW SRP fraction can make... 

DOM is derived from autochthonous sources such as phytoplankton and photosynthetic bacteria (16) at Big Soda Lake near Fallon, Nevada. This lake is alkaline (pH 9.7) and chemically stratified. It contains DOC concentrations as high as 60 mg/L and dissolved salt concentrations as high as 88,000 mg/ L (17). The DOM in this lake is colorless. The fulvic acid fraction was isolated by adsorption chromatography (Amberlite XAD-8 resin) (18) and by zeo-trophic distillation of water from N,N-dimethylformamide (19). Average molecular model synthesis was achieved in a manner similar to that used for fulvic acid from the Suwannee River. The characterization data are presented in Table I and the structural model is presented in Structure 2. 

C18 solid-phase extraction is used to fractionate polyphenolics for their identification and characterization. This technique can eliminate interfering chemicals from crude extracts and produce desirable results for HPLC or other analytical procedures. To obtain a sufficient volume for all analyses, several separations by solid-phase extraction may be performed. The individual fractions need to be combined and dissolved in solvents appropriate for HPLC analysis. In Basic Protocol 2, the application of a current of nitrogen gas for the removal of water from the C18 cartridge is an important step in the selective fractionation of polyphenolics into non-anthocy-anin and anthocyanin fractions. After the collection of non-anthocyanin polyphenolics, no additional work is necessary to elute anthocyanins bound to the C18 solid phase if anthocyanins are not to be determined. 

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